NMDA receptor subunits in the adult rat hippocampus undergo similar changes after 5 minutes in an open field and after LTP induction.

NMDA receptor subunits change during development and their synaptic expression is modified rapidly after synaptic plasticity induction in hippocampal slices. However, there is scarce information on subunits expression after synaptic plasticity induction or memory acquisition, particularly in adults. GluN1, GluN2A and GluN2B NMDA receptor subunits were assessed by western blot in 1) adult rats that had explored an open field (OF) for 5 minutes, a time sufficient to induce habituation, 2) mature rat hippocampal neuron cultures depolarized by KCl and 3) hippocampal slices from adult rats where long term potentiation (LTP) was induced by theta-burst stimulation (TBS). GluN1 and GluN2A, though not GluN2B, were significantly higher 70 minutes--but not 30 minutes--after a 5 minutes session in an OF. GluN1 and GluN2A total immunofluorescence and puncta in neurites increased in cultures, as evaluated 70 minutes after KCl stimulation. Similar changes were found in hippocampal slices 70 minutes after LTP induction. To start to explore underlying mechanisms, hippocampal slices were treated either with cycloheximide (a translation inhibitor) or actinomycin D (a transcription inhibitor) during electrophysiological assays. It was corroborated that translation was necessary for LTP induction and expression. The rise in GluN1 depends on transcription and translation, while the increase in GluN2A appears to mainly depend on translation, though a contribution of some remaining transcriptional activity during actinomycin D treatment could not be rouled out. LTP effective induction was required for the subunits to increase. Although in the three models same subunits suffered modifications in the same direction, within an apparently similar temporal course, further investigation is required to reveal if they are related processes and to find out whether they are causally related with synaptic plasticity, learning and memory

Glypican-3 (GPC3) inhibits metastasis development promoting dormancy in breast cancer cells by p38 MAPK pathway activation

GPC3 is a proteoglycan involved in the control of proliferation and survival, which has been linked to several tumor types. In this respect, we previously demonstrated that normal breast tissues exhibit high levels of GPC3, while its expression is diminished in tumors. However, the role of the GPC3 downregulation in breast cancer progression and its molecular and cellular operational machineries are not fully understood. In this study we showed that GPC3 reverts the epithelial-to-mesenchymal transition (EMT) underwent by mammary tumor cells, blocks metastatic spread and induces dormancy at secondary site. Using genetically modified murine breast cancer cell sublines, we demonstrated that the phospho-Erk/phospho-p38 ratio is lower in GPC3 reexpressing cells, while p21, p27 and SOX2 levels are higher, suggesting a dormant phenotype. In vivo metastasis assays confirmed that GPC3 reexpressing cells reduce their metastatic ability. Interestingly, the presence of dormant cells was evidenced in the lungs of inoculated mice. Dormant cells could reactivate their proliferative capacity, remain viable as well as tumorigenic, but they reentered in dormancy upon reaching secondary site. We also proved that GPC3 inhibits metastasis through p38 pathway activation. The in vivo inhibition of p38 induced an increase in cell invasion of GPC3 reexpressing orthotropic tumors as well as in spontaneous and experimental metastatic dissemination. In conclusion, our study shows that GPC3 returns mesenchymal-like breast cancer cells to an epithelial phenotype, impairs in vivo metastasis and induces tumor dormancy through p38 MAPK signaling activation. These results help to identify genetic determinants of dormancy and suggest the translational potential of research focusing in GPC3.Fil: Guereño, Macarena. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Delgado Pastore, Magali. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Lugones, Ana Clara. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Cercato, Magalí Cecilia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Todaro, Laura Beatriz. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Urtreger, Alejandro Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Peters, María Giselle. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentin

NMDAR subunits immunofluorescence in mature hipocampal neuron cultures stimulated by KCl.

<p><b><i>A.</i></b> Quantification of NMDAR subunit puncta at dendrites (n = 100 neurites/culture). A significant increase in GluN1 and GluN2A puncta was observed at 30 and 70 minutes after KCl stimulation (** p<0.05, *** p<0.001, Kruskal-Wallis test followed by Dunn’s Multiple Comparison Post-Test). <i>Insert on the top of each bar</i>: representative dendrite for each condition (bar: 2 µm). <b><i>B.</i></b> Total fluorescence quantification 30 and 70 minutes after KCl stimulation. There were significant increases in GluN1 and GluN2A 70 minutes after stimulation. There were no significant changes in GluN2B total immunofluorescence (* p<0.05, *** p<0.001, ONE WAY ANOVA, Dunnet Post-Test). <i>Right:</i> representative neurons for each condition.</p

NMDAR subunits change after LTP induction and expression.

<p><b><i>A.</i></b> Evoked fEPSPs normalized slopes from fresh hippocampal slices corresponding to the first pulse of paired stimulation, before and after TBS (arrow). Plots represent the average of three independent experiments over 90 minutes of recording (n = 6 for each group). <i>Insert on top:</i> average traces of 10 individual recordings from a +TBS+LTP and a +TBS-LTP slices (black: 5 minutes before TBS; grey: 5 last minutes of recording). <b><i>B.</i></b> WB band densities quantification of samples from same slices that in <b>A.</b> A significant increase was only observed for +TBS+LTP slices (** p<0.01; *** p<0.001 ONE WAY ANOVA, Dunnet Post-Test; n = 6 for each group). <i>Insert on top:</i> (from left to right): representative GluN1 and GAPDH WB bands from: a −TBS slice, a +TBS-LTP slice and a +TBS+LTP slice. <b><i>C.</i></b> Evoked fEPSPs slopes corresponding to the first pulse of the paired stimulation before and after TBS (arrow). Plots represent the average of fEPSPs slopes over 50 and 90 minutes of recording, respectively (n = 6 for each group). <i>Right</i>: average traces of 10 individual recordings from a LTP-slice after 30 and 70 minutes TBS (black: 5 minutes before TBS; grey: 5 last minutes of recording). <b><i>D.</i></b> NMDAR subunits quantification by WB. Samples analyzed: slices used in <b>C</b>. (processed 30 or 70 minutes after TBS) and in −TBS slices (Control). Analysis of WB bands showed a significant increase in GluN1 and GluN2A level for the 70 minutes group in three independent experiments (* p<0,05; *** p<0,001 ONE WAY ANOVA-Dunnet Test). <i>Insert on top:</i> Representative WB bands for GluN1, GluN2A and GluN2B NMDAR subunits and GAPDH (internal control).</p

NMDAR subunits changes after OF habituation. <i>A.</i>

<p>Habituation to the OF of rats exposed to a 5 minutes OF session (n = 16). Graphs show number of crossings (left panel) and rearings (right panel) <i>per</i> minute (bars indicates median with interquartile ranges). Crossings decreased significantly after 3 minutes, while rearings were only significantly decreased in the fifth minute. *** p<0.0001, ** p<0.01 by Friedman test followed by Dunn’s Multiple Comparison Test. <b><i>B.</i></b> Total crossings from rats exposed to the OF for 1 or 5 minutes (Training) and tested for STM 40 minutes later (n = 12) or LTM 24 h later (n = 16). There were significant differences in total number of crossings in the second session compared to the first, only in rats which spent 5 minutes in the OF in the training session, for STM (* p<0.05) as well as for LTM (*** p<0.0001) (Mann Whitney test). <b><i>C.</i></b> NMDAR subunits in the hippocampus of rats after OF exposure. Four groups of rats were analyzed: rats as in <b><i>A,</i></b> which were sacrificed at 0, 30 and 70 minutes after the task (5′-0′, 5′–30′ and 5′–70′ groups); and rats exposed for 1 minute to the OF, sacrificed 70 minutes later (1′–70′ group). WB analysis showed about a one fold increase in GluN1 and GluN2A level for <i>5′–70′</i> group, in 3 independent experiments (* p<0.05, ONE WAY ANOVA, Newman-Keuls Multiple Comparison Post-Test). <i>Insert on top:</i> representative WB bands for GluN1, GluN2A and GluN2B NMDAR subunits and GAPDH (internal control). <b><i>D</i></b>. NMDAR subunits analysis in the hippocampus of rats after two OF sessions. 4 groups of rats were analyzed: rats exposed to the OF 5 minutes and sacrificed immediately (5′-0′), 70 minutes (5′–70′), 24 h later (5′–24 h), or tested in the OF and sacrificed 70 minutes later (70′ postest-TE). * p<0.05 ONE WAY ANOVA, Dunnett’s Post-Test. <i>Insert on top:</i> representative WB bands for GluN1 and GluN2A NMDAR subunits and GAPDH (internal control).</p

NMDAR subunits modification in hippocampal slices after transcription or translation inhibition during LTP induction. <i>A.</i>

<p><i> Left:</i> Normalized slopes of evoked fEPSPs recorded as those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055244#pone-0055244-g003" target="_blank">Figure 3</a>, corresponding to the first pulse of the paired stimulation applied before and after TBS (arrow). Plots represent the average of five independent experiments over 90 minutes of recording (n = 5 for each group). <i>Black line</i>: drug perfusion. <i>Insert on top</i>: average traces of 10 individual recordings from a control slice and slices treated with ActD or CHX (black: 5 minutes before TBS; grey: 5 last minutes of recording). <i>Right:</i> Bars represent averages of normalized first pulse slopes of the 5 last minutes of recording for each group. LTP induction was blocked by 40 µg/ml CHX treatment (* p<0.05, one sample t test) compared to basal transmission (line referred to 1 in the graph). <b><i>B.</i></b> NMDAR subunits were evaluated by WB in same slices that in <b>A.</b> CHX treatment blocked GluN1 and GluN2A increase, while 50 µg/ml ActD only blocked GluN1 increase (* p<0,05 ONE WAY ANOVA, Dunnet Post-Test; n = 5 for each group). <i>Insert on top:</i> (from left to right): Representative GluN1 and GluN2A WB bands of +TBS+LTP slices (control), CHX and ActD +TBS+LTP slices treated slices. <b><i>C.</i></b> Table indicates mean ± SEM for GluN1/GAPDH (first row) or GluN2A/GAPDH (second row) in +TBS-LTP slices (n = 6) and +TBS+LTP slices without any drug treatment (Control in <b>B,</b> n = 9), or treated with ActD (n = 5) or CHX (n = 5) (*** p<0.0001; ONE WAY ANOVA - Newman Keuls Test).</p